Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.
In the last few decades, technical advances in altering the genes of organisms have led to many discoveries about how genes work. For example, it is now possible to add a specific DNA sequence to a gene so that the protein it makes will carry a ‘tag’ that enables us to track it in cells. One such tag is called green fluorescent protein (GFP) and it is often used to study other proteins in living cells because it produces green fluorescence that can be detected under a microscope.
It is labor intensive to add tags to individual genes, so this limits the number of proteins that can be studied in this way. In 2011, researchers developed a new method that can easily tag many genes in fruit flies. It makes use of small sections of DNA called transposons, which are able to move around the genome by ‘cutting’ themselves out of one location and ‘pasting’ themselves in somewhere else.
The researchers used a transposon called Minos, which is naturally found in fruit flies. When Minos inserts into a gene, it often disrupts the gene and stops it from working. However, the researchers could swap the inserted transposon for a gene encoding GFP by making use of a natural process that rearranges DNA in cells. This resulted in the protein encoded by the gene containing GFP and so it can be detected under a microscope. This method allowed the researchers to create a collection of fly lines that have the GFP tag on many different proteins.
Now, Nagarkar-Jaiswal et al. have greatly expanded this initial collection. More than 75% of GFP-tagged proteins worked normally and the flies producing these altered proteins remain healthy. It is possible to use a technique called RNA interference against the GFP to lower the production of the tagged proteins. Moreover, Nagarkar-Jaiswal et al. show that it is also possible to degrade the tagged proteins so that less protein is present. The removal of proteins is reversible and can be done in specific tissues during any phase in fly development. These techniques allow researchers to directly associate the loss of the protein with the consequences for the fly.
This collection of fruit fly lines is a useful resource that can help us understand how genes work. The method for tagging the proteins could also be modified to work in other animals.